PROJECT SUMMARY Although the health benefits of physical activity in prevention and mitigation of many chronic diseases have been documented for decades, the molecular mechanisms mediating these health benefits are still poorly understood. To understand the adaptive response of physical activity at the molecular level, it is essential to comprehensively characterize the dynamic changes in protein expression and post-translational modifications (PTMs) in tissues and circulating biofluids. The overall objective of the PNNL Proteomics Chemical Analysis Center for MoTrPAC (ProMoTr) is to support the consortium by providing comprehensive discovery and subsequent targeted verification analyses of circulating protein factors in blood and associated PTMs in multiple tissue types collected by the Pre-clinical Animal Study Sites and Clinical Centers of MoTrPAC. To develop a `molecular map' of transducers that provide the impacts of physical activity in humans, the ProMoTr's integrative proteomics analysis plan has two major foci. First, we will focus on the circulating secreted factors found in blood plasma and tissues based on the known cross-talk between and within tissues resulting from physical activity. Second, we will investigate the tissue- and cellular- level response to physical activity by assessing intracellular signaling as mediated by protein PTMs, including protein phosphorylation, reversible redox modifications, and lysine acetylation and acylation. The Specific Aims of ProMoTr are: 1) to provide the Proteomics Chemical Analysis Element to discover and verify circulating factors and intracellular molecular transducers; 2) to provide the Bioinformatics Element to fully support the needs of ProMoTr in statistical design, data processing, analysis, interpretation, and dissemination; 3) to provide the Administrative Element to oversee internal activities and to provide high-level collaborative interactions with MoTrPAC centers and the broader consortium. The feasibility of performing high quality reproducible discovery and verification analyses on the scale of tens of thousands of tissue samples is built upon PNNL's record of accomplishment of large-scale integrative proteomics programs. The PNNL foundation includes refined quality control, large-scale computation, and dissemination capabilities, our recognized expertise in the development and deployment of cutting-edge mass spectrometry-based technologies, and our capabilities for studying protein PTMs that are closely linked with energy metabolism.